Promotive Effect Of Magnesium Ions On Biological Behaviours Of Osteoblasts And The Underlying Mechanism

Magnesium and its alloys, which have been used for biodegradable metallic materials biomedical for the last 200 years,have received more attention due to their low elastic modulus, biodegradability, low toxicity and bioactivity. However, the mechanisms underlying the stimulatory effect of magnesium on osteogenesis remain elusive. Some researches find that the corrosion products of magnesium implants lead to an increase in the magnesium ion(Mg2+) concentration around the implant, and this effect has been shown to remarkably enhance new bone formation around the implant. And other researches find that adding Mg2+ to scaffolds has been demonstrated to remarkably enhance new bone formation. Recently, a growing body of literature has indicated that PI3K/Akt signalling pathway is critical for both proliferation and differentiation of osteoblasts. Therefore, we postulate that the stimulatory effect of magnesium on osteogenesis may result from the Mg2+-induced activation of the PI3K/Akt signallingpathway in osteoblasts. The elucidation of the stimulatory effect of Mg2+ on biological behaviours of osteoblasts may provide a good theoretical reference for the clinical application of Mg-based metallic materials in the future.Objective: To investigate the effect of different concentrations of Mg2+ on biological behaviours of osteoblasts, and to explore the mechanisms underlying the stimulatory effect of Mg-based metallic materials on osteogenesis.Methods: 1. A 50 m M Mg Cl2 solution was prepared by dissolving Mg Cl2 in Dulbecco’s modified Eagle’s medium(DMEM; Gibco). Subsequently, Mg2+-containing culture medium containing 6, 10 and 18 m M Mg2+ was prepared by diluting the 50 m M Mg Cl2 solution directly, by sterile filtering through a membrane filter. DMEM culture medium(Mg2+ concentration: 0.8 m M; Gibco) was used as the control group. 2. Primary rat calvarial osteoblasts were isolated and extracted by digestion of the calvarial bone of 1-day-old Sprague-Dawley rats under aseptic condition. Passage 2-3 osteoblasts were seeded into culture medium at a density of 5 × 104 cells/ml and were randomised to culture with one of the following groups:(1) DMEM culture medium(DMEM);(2) DMEM culture medium containing 6 m M Mg2+(6 m M);(3) DMEM culture medium containing 10 m M Mg2+(10 m M);(4) DMEM culture medium containing 18 m M Mg2+(18 m M);(5) 10 m M Mg2+ + wortmannin(10 m M + W; W = wortmannin, a PI3K/Akt-specific inhibitor). The morphological changes of cultured cells were observed. 3. The cell adhesion of osteoblasts was measured by cytoskeleton staining assay. The viability of osteoblasts was measured by MTT assay. The osteogenic differentiation of osteoblasts was measured by ALP activity, Alizarin red staining and expression of osteogenic differentiation-related genes. Western blot was used to determine the activation of PI3K/Akt signaling pathway in different Mg2+ concentration. Semi-quantitative analysis was used to determine the level of p-Akt expressionin in different groups. We further identified whether the PI3 K inhibitor wortmannin influenced the stimulatory effect of 10m M Mg2+ on biological behaviours of osteoblasts.Results: The groups cultured with 6 and 10 m M Mg2+ showed enhanced cell adhesion, cell viability, ALP activity, extracellular matrix mineralization and expression of osteogenic differentiation-related genes(p<0.05), while 18 m M Mg2+ group showed the opposite effect(p<0.05). Western blot showed that phosphorylation level of Akt in 6 m M and 10 m M Mg2+ was significantly increased compared with the control group, while the 18 m M group exhibited an obvious decrease in the phosphorylation level of Akt compared with the control group(p<0.05). However, the enhancing function of 10 m M Mg2+ was reversed by co-treatment with wortmannin(p < 0.05). A possible role of the PI3K/Akt signalling pathway in the biological behaviour of osteoblasts cultured in 10 m M Mg2+ was further verified. Results showed that the aforementioned beneficial effects afforded by 10 m M Mg2+ on cell adhesion, cell viability, ALP activity, extracellular matrix mineralization and expression of osteogenic differentiation-related genes were abolished by blocking the PI3K/Akt signaling pathway with addition of wortmannin, which further identified the stimulatory effect of PI3K/Akt signalling pathway on biological behaviours of osteoblasts.Conclusions: The biological behaviours of osteoblasts were promoted by 6 and 10 m M Mg2+, while inhibited by the 18 m M Mg2+. The promotive effect of 10 m M Mg2+ on biological behaviours of osteoblasts attributed to the activation of the PI3K/Akt signaling pathway. This study could promisingly provide a good theoretical reference for the clinical application of Mg-based metallic materials in the future.